CN110218748B - Method for producing grease by using microalgae at low cost - Google Patents
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Abstract
The invention provides a method for producing grease by using microalgae at low cost, belonging to the technical field of microalgae biological treatment. The method provided by the invention comprises the steps of inoculating microalgae into saline-alkali soil leacheate, culturing for 25-30 days at 20-30 ℃, and then separating oil from a culture solution. The saline-alkali soil leacheate is used for culturing the microalgae, contains a large amount of nutrients and trace elements necessary for the growth of the microalgae, and is favorable for the accumulation of algae lipids due to proper salinity. The method provided by the invention reduces the microalgae cultivation cost, purifies the saline-alkali soil leacheate while obtaining high-yield biomass, and improves the wastewater recycling efficiency. In addition, the method provided by the invention utilizes the saline-alkali soil leacheate to culture microalgae, can convert biomass accumulated grease at low cost, and is suitable for popularization and application.
Description
Technical Field
The invention belongs to the technical field of microalgae biological treatment, and particularly relates to a method for producing oil at low cost by using microalgae.
Background
The salinization of soil is a worldwide environmental problem, and saline-alkali soil has large area and great treatment difficulty. Saline-alkali soil, as a potential land resource, has the potential of becoming a high-quality agricultural land or grassland, and can bring huge benefits if reasonably developed and utilized. According to the data of the department of agriculture in china, it is shown that there are about 34 thousand square kilometers of saline land in china, of which about 12.4 thousand square kilometers of land can be further used for agricultural production after remediation. Soil salinization not only affects soil properties, reduces soil productivity, but also causes ecosystem changes such as growth and distribution of plants, eventually leading to deterioration of grasslands. At present, the improvement of saline-alkali soil has received wide attention. The known measures for improving saline-alkali soil mainly include water conservancy measures, physical measures, chemical measures and biological measures. The saline-alkali land treatment in the world generally adopts an irrigation leaching method, however, the method needs a large amount of agricultural irrigation water resources, discharges a large amount of leaching liquid containing plant nutrients such as nitrogen, phosphorus and potassium, and the saline-alkali wastewater usually needs to be further treated and discharged, so that the treatment cost is increased.
Microalgae is an ideal raw material for third-generation bioenergy because of the advantages of no cultivated land occupation, short growth cycle, high oil content and the like, has strong environmental adaptability, can accumulate various high-value metabolites including protein, polysaccharide, pigment, fatty acid and the like through photosynthesis, and has recently attracted attention in the fields of medicine, food, resource and energy development. The preparation of biodiesel and other high-value products by using microalgae is always a global research hotspot, but the problems of difficult culture, high production cost and the like are serious, and the popularization and application of the microalgae in the production of grease are limited.
Disclosure of Invention
In view of the problems of the background art, it is an object of the present invention to provide a method for producing an oil or fat at a low cost using microalgae.
The invention provides a method for producing grease by using microalgae, which comprises the steps of inoculating the microalgae into saline-alkali soil leacheate, culturing for 25-30 days at 20-30 ℃, and then separating the culture solution to obtain the grease.
Preferably, the microalgae comprise chlorella and/or scenedesmus.
Preferably, the saline-alkali soil leacheate is obtained by leaching saline-alkali soil with reclaimed water.
Preferably, the salinity of the saline-alkali soil leacheate is 0.14-0.6%.
Preferably, the saline-alkali soil leacheate is subjected to filtration and autoclaving treatment before use; the aperture of the filter membrane for filtration is 0.4-0.5 μm; the temperature of the high-pressure sterilization is 118-125 ℃, and the time of the high-pressure sterilization is 20-40 min.
Preferably, the microalgae has a density of 2 × 10 upon inoculation5～2×106one/mL.
Preferably, the light-dark ratio of the culture is 13-15 h: 9-11 h.
Preferably, the light intensity during the light culture is 50 to 70. mu. mol phosns. m-2·s-1。
Preferably, the microalgae is chlorella HQ; the density of the microalgae is 2 multiplied by 10 when being inoculated5～5×105Per mL; the culture temperature is 25-26 ℃.
The invention also provides the grease prepared by the method.
Has the advantages that: the invention provides a method for producing grease by using microalgae, which comprises the steps of inoculating the microalgae into saline-alkali soil leacheate, culturing for 25-30 days at 20-30 ℃, and then separating the culture solution to obtain the grease. The saline-alkali soil leacheate is used for culturing the microalgae, contains a large amount of nutrients and trace elements necessary for the growth of the microalgae, and is favorable for the accumulation of algae lipids due to proper salinity. The method provided by the invention reduces the microalgae cultivation cost, purifies the saline-alkali soil leacheate while obtaining high-yield biomass, and improves the wastewater recycling efficiency. In addition, the method provided by the invention utilizes the saline-alkali soil leacheate to culture microalgae for converting biomass accumulated grease, has low cost and is suitable for popularization and application.
Biological preservation information description
The chlorella HQ is preserved in the China general microbiological culture Collection center in 2013, 5 and 7 months, and the preservation number of the chlorella HQ is CGMCC No. 7601;
scenedesmus LX1, which is preserved in China general microbiological culture Collection center at 23.4.2009, with the collection number of CGMCC No. 3036.
Drawings
FIG. 1 is a graph showing the growth curves of three oil-producing microalgae according to example 1 in a saline-alkali soil leacheate (Shandong);
FIG. 2 shows the oil accumulation characteristics of three oil-producing microalgae according to example 1 in the leaching solution (Shandong) of saline-alkali soil;
FIG. 3 is a growth curve of three oil-producing microalgae according to example 2 of the present invention in saline-alkali soil leacheate (inner Mongolia);
fig. 4 shows the oil accumulation characteristics of the three oil-producing microalgae in example 2 of the present invention in saline-alkali soil leacheate (inner Mongolia).
Detailed Description
The invention provides a method for producing grease by using microalgae, which comprises the steps of inoculating the microalgae into saline-alkali soil leacheate, culturing for 25-30 days at 20-30 ℃, and then separating the culture solution to obtain the grease.
The invention firstly inoculates microalgae into the saline-alkali soil leacheate.
In the present invention, the microalgae preferably include chlorella and/or scenedesmus, the chlorella includes chlorella HQ (obtained by separation at the prophase of the subject group) and chlorella vulgaris (purchased from fresh water algae seed bank of the chinese academy of sciences), preferably chlorella HQ, and the scenedesmus preferably is scenedesmus LX1 (provided by the simulation and pollution control laboratory of the university of qinghua); in the most preferred scheme of the invention, chlorella HQ is used for producing the grease.
In the invention, the saline-alkali soil leacheate is preferably obtained by leaching saline-alkali soil with reclaimed water. The reclaimed water is preferably terminal water of a sewage treatment plant; in a more specific embodiment of the invention, the reclaimed water is terminal water of a Beijing-city Beizhou river sewage treatment plant, and the effluent water quality standard is the B-level discharge standard in the Beijing-city local standard water pollution comprehensive discharge standard (DB 11/307-2013). The source of the saline-alkali soil is not particularly limited in the present invention. In the invention, the salinity of the saline-alkali soil is preferably 0.14-0.6%, more preferably 0.2-0.5%, and more preferably 0.25-0.4%. The pH value of the saline-alkali soil is preferably 7.5-8.5.
In the invention, the saline-alkali soil leacheate is preferably subjected to filtration and autoclaving treatment before use; the pore diameter of the filter membrane for filtration is preferably 0.4-0.5 μm, and more preferably 0.45 μm; the temperature of the high-pressure sterilization is preferably 118-125 ℃, and more preferably 121 ℃; the time for autoclaving is preferably 20-40 min, and more preferably 30 min.
In the present invention, the density of the microalgae upon inoculation is preferably 2 × 105～2×106one/mL, more preferably 2X 105～5×105one/mL.
The invention inoculates microalgae into saline-alkali soil leacheate and then carries out culture.
In the invention, the temperature of the culture is preferably 20-30 ℃, and more preferably 25-26 ℃. The light-dark ratio of the culture is preferably 13-15 h: 9-11 h, more preferably 14 h: and (5) 10 h. In the case of light culture, the light intensity of the light culture is preferably 50 to 70. mu. mol phosns. m-2·s-1More preferably 60. mu. mol of photons. m-2·s-1. The incubation time is preferably 25-30 d, and more preferably 28 d.
After the culture is finished, the invention obtains the oil produced by the microalgae in the culture solution. Through separation and determination, the highest lipid yield of the microalgae cultured by the saline-alkali soil leacheate can reach 69.90 mg/L.
The method provided by the invention can save carbon sources, nitrogen sources and other trace elements, and reduce the cost of microalgae culture; the saline-alkali soil leacheate is purified, the recycling efficiency of the leacheate is improved, energy is saved, and the environment is protected.
The three oil-producing microalgae can well grow in the saline-alkali soil leacheate, a new idea is provided for the treatment of the saline-alkali soil leacheate, and nutrient substances are converted into microalgae biomass; the possible action mechanism is that the carbon source and the nitrogen source in the saline-alkali soil leacheate are absorbed and utilized by the microalgae, wherein the inorganic carbon source is mainly utilized. The method screens out the best chlorella HQ growing in the saline-alkali soil leacheate, and the yield of the microalgae grease is high.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
The reclaimed water in the following examples was obtained from the terminal water of a sewage treatment plant of beijing, beixianhe; saline-alkali soil is respectively taken from eastern Shandong Ying and inner Mongolia autonomous region and local Mei Teh Zhan Qizhen city of Haote city.
The method for measuring the algae density in the following examples adopts a cell counting method, namely, a certain amount of algae liquid is taken, shaken up, the cell density of algae in the culture solution is monitored by a blood counting plate under an optical microscope, the algae density is measured every two days, and a microalgae growth curve is drawn.
The steps for determining the microalgae oil in the following examples are as follows: placing 40mL of algae solution in a 50mL centrifuge tube, centrifuging at 4 deg.C at 10000rpm for 10min, and pouring out supernatant until the volume of algae solution is 0.8 mL. To the concentrated solution were added 1mL of chloroform and 2mL of methanol in this order, followed by thoroughly mixing, further adding 1mL of chloroform and 1mL of purified water, thoroughly shaking again, mixing, and then centrifuging (4 ℃, 4000 rpm. times.10 min). The lower organic phase was transferred to a dry weighed glass digestion tube with a pipette and nitrogen was blown to constant weight. And weighing the weight of the glass tube again, wherein the difference between the front weight and the rear weight is the total grease mass.
Example 1
(1) Collecting saline-alkali soil samples: randomly picking 10 sampling points on saline-alkali soil land in east Shandong region according to a snake sampling method. Sampling the upper layer (0-20cm), the middle layer (20-40cm) and the lower layer (40-60cm) of the soil at each sampling point by using a professional sampler to obtain upper, middle and lower layer soil samples. And then, naturally drying, grinding and sieving each layer of soil sample by a 3mm sieve, uniformly mixing, and storing by layers.
(2) And putting the retrieved upper, middle and lower saline-alkali soil samples into a leaching device (the specification is 100 multiplied by 92 multiplied by 70mm) from bottom to top according to the sequence of the lower layer, the middle layer and the upper layer, and leaching with regenerated water to obtain leaching solution. Filtering the eluate with 0.45 μm water-washing filter membrane, sterilizing with 121 deg.C high pressure steam for 30min, and cooling to room temperature to obtain culture medium. The pH and salinity of the medium were determined to be 7.73 and 0.28%.
(3) Dividing the culture medium obtained in the step (2) into three parts, and respectively inoculating chlorella vulgaris, chlorella HQ and scenedesmus LX 1. The initial inoculation densities were all 2X 105one/mL. At a temperature of 25 ℃, the light-dark ratio is 14 h: 10h, the illumination intensity is 60 mu mol photons.m-2·s-1Culturing for 25 days.
(4) In the culture process, detecting the density of algae cells in the culture solution by using a blood counting plate, and drawing a microalgae growth curve once every two days; and after the culture is finished, measuring the oil content of the microalgae.
The results are shown in FIGS. 1 to 2.
FIG. 1 is a graph showing the growth curves of three oil-producing microalgae in saline-alkali soil leacheate (Shandong). As can be seen from fig. 1: in the initial stage of culture, the growth rate of chlorella HQ and chlorella vulgaris is relatively high, while the growth rate of scenedesmus LX1 is relatively low. After 25 days of culture, the density of Chlorella HQ, Chlorella vulgaris and Scenedesmus LX1 reaches 1.65 × 1071.41X 10 units/mL7one/mL and 1.22X 107one/mL. The method shows that the density of the chlorella HQ obtained after the chlorella HQ is cultured in the saline-alkali soil leacheate for 25 days is the highest.
FIG. 2 shows the oil accumulation characteristics of three oil-producing microalgae in saline-alkali soil leacheate (Shandong). As can be seen from fig. 2: after the three microalgae are cultured in the saline-alkali soil leacheate for 25 days, the lipid yield of the chlorella HQ is 69.90mg/L at most, and is about twice of that of chlorella vulgaris and Scenedesmus LX 1. The method shows that the chlorella HQ can accumulate a large amount of grease in the saline-alkali soil leacheate.
Example 2
(1) Collecting saline-alkali soil samples: according to a snake-shaped sampling method, 10 sampling points are randomly picked on saline-alkali soil land in an inner Mongolia area. Sampling the upper layer (0-20cm), the middle layer (20-40cm) and the lower layer (40-60cm) of the soil at each sampling point by using a professional sampler to obtain upper, middle and lower layer soil samples. And then, naturally drying, grinding and sieving each layer of soil sample by a 3mm sieve, uniformly mixing, and storing by layers.
(2) And putting the retrieved upper, middle and lower saline-alkali soil samples into a leaching device (the specification is 100 multiplied by 92 multiplied by 70mm) from bottom to top according to the sequence of the lower layer, the middle layer and the upper layer, and leaching with regenerated water to obtain leaching solution. Filtering the eluate with 0.45 μm water-washing filter membrane, sterilizing with 121 deg.C high pressure steam for 30min, and cooling to room temperature to obtain culture medium. The pH of the medium was determined to be 8.50 and the salinity was determined to be 0.38%.
(3) Dividing the culture medium obtained in the step (2) into three parts, and respectively inoculating chlorella vulgaris, chlorella HQ and scenedesmus LX 1. The initial inoculation densities were all 2X 105one/mL. At a temperature of 25 ℃, the light-dark ratio is 14 h: 10h, the illumination intensity is 60 mu mol photons.m-2·s-1Under the conditions of (3) for 28 d.
(4) In the culture process, detecting the density of algae cells in the culture solution by using a blood counting plate, and drawing a microalgae growth curve once every two days; and after the culture is finished, measuring the oil content of the microalgae.
The results are shown in FIGS. 3 to 4.
FIG. 3 is a growth curve of three oil-producing microalgae in saline-alkali soil leacheate (inner Mongolia). As can be seen from fig. 3: after culturing for 28 days, the density of Chlorella HQ, Chlorella vulgaris and Scenedesmus LX1 reaches 1.16 × 1070.75X 10 cells/mL7one/mL and 0.63X 107The chlorella HQ has better growth condition per mL.
Fig. 4 shows the oil accumulation characteristics of three oil-producing microalgae in saline-alkali soil leacheate (inner Mongolia). As can be seen from fig. 4: after the three microalgae are cultured in the saline-alkali soil leacheate for 28 days, the lipid yield of the chlorella HQ is the highest and is 54.52 mg/L.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (1)
1. A method for producing grease by using microalgae is characterized in that the microalgae is inoculated into saline-alkali soil leacheate and cultured for 25-30 days at 25 ℃, and then grease is obtained by separating from a culture solution;
the microalgae is chlorella HQ;
the light-dark ratio of the culture is 14 h: 10 h;
the saline-alkali soil leacheate is obtained by leaching saline-alkali soil with reclaimed water;
the salinity of the saline-alkali soil leacheate is 0.14-0.6%;
the saline-alkali soil leacheate is subjected to filtration and autoclaving treatment before use; the aperture of the filter membrane for filtration is 0.4-0.5 μm; the temperature of the high-pressure sterilization is 118-125 ℃, and the time of the high-pressure sterilization is 20-40 min;
the density of the microalgae is 2 multiplied by 10 when being inoculated5；
The illumination intensity during the light culture is 50-70 mu mol phosns.m-2·s-1；
The preservation number of the chlorella HQ is CGMCC No. 7601.
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| CN103571754B (en) * | 2013-05-09 | 2016-05-18 | 北京林业大学 | One cultural method of chlorella Chlorella sp.HQ and the application of purification of water quality produce oil thereof |
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